Nucleic acid libraries provide tremendous opportunities for the selection of novel ligands and catalysts since the polymerase chain reaction, PCR, allows for the synthesis and selection of libraries containing more than 10.sup.14 different molecules. There are now many examples of nucleic acids that have been selected to bind proteins and small molecules and to catalyze a limited set of reactions (S. E. Osborne, A. D. Ellington, Chem. Rev. (Washington, D.C.) 1997, 97, 349-370; M. Famulok, J. W. Szostak, Angew. Chem. 1992, 104, 1001-11; Angew. Chem.Int. Ed. Engl. 1992, 31, 979-88; L. Gold, B. Polisky, O. Uhlenbeck, M. Yarus, Annu. Rev. Biochem. 1995, 64, 763-97; R. R. Breaker, Chem. Rev. (Washington, D.C.) 1997, 97, 371-390; R. R. Breaker, Curr. Opin. Chem. Biol. 1997, 1, 26-31; J. R. Lorsch, J. W. Szostak, Acc. Chem. Res. 1996, 29, 103-10; R. R. Breaker, G. F. Joyce, Chem. Biol. 1994, 1, 223-9; B. Cuenoud, J. W. Szostak, Nature (London) 1995, 375, 611-14; R. R. Breaker, G. F. Joyce, Chem. Biol. 1995, 2, 655-60; C. R. Geyer, D. Sen, Chem. Biol. 1997, 4, 579-593; S. W. Santoro, J. G. F., Proc. Natl. Acad Sci. U.S.A. 1997, 94, 4262-4266; P. Burgstaller, M. Famulok, Angew. Chem. 1995,107,1303-06; Angew. Chem. Int. Ed. Engl. 1995, 34, 1189-92; D. Faulhammer, M. Famulok, Angew. Chem. 1996, 108, 2984-88; Angew. Chem. Int. Ed. Engl. 1996,35,2837-2841; D. Faulhammer, M. Famulok, J. Mol. Biol. 1997, 269, 188-202; Y. Li, D. Sen, Nat. Struct. Biol. 1996, 3, 743-747; J. Burmeister, G. von Kiedrowski, A. D. Ellington, Angew. Chem. 1997, 109, 1379-81; Angew. Chem. Int. Ed. Engl. 1997,36, 1321-1324; N. Carni, L. A. Shultz, R. R. Breaker, Chem. Biol. 1996, 3, 1039-1046; N. Carmi, H. R. Balkhi, R. R. Breaker, Proc. Natl. Acad Sci. U.S.A. 1998, 95, 2233-2237).
The catalytic and mechanistic scope of nucleic acids is limited since the natural nucleotide monomers possess minimal functionality compared to the repertoire available to Nature's dominant catalytic biopolymers, proteins. In recognition of this shortcoming, much attention has been focused on the development of functionalized nucleotides suitable for in vitro selection with the hope of increasing the potential of nucleic acids for binding and catalysis (B. E. Eaton, W. A. Pieken, Annu. Rev. Biochem. 1995, 64, 837-63; B. E. Eaton, Curr. Opin. Chem. Biol. 1997, 1, 10-16; G. J. Crouch, B. E. Eaton, Nucleosides Nucleotides 1994, 13 939-44; T. M. Dewey, A. Mundt, G. J. Crouch, M. C. Zyzniewski, B. E. Eaton, J. Am. Chem. Soc. 1995, 117, 8474-5; T. M. Dewey, M. C. Zyzniewski, B. E. Eaton, Nucleosides Nucleotides 1996, 15, 1611-1617; C. Tu, C. Keane, B. E. Eaton, Nucleosides Nucleotides 1995, 14, 1631-8; P. A. Limbach, P. F. Crain, J. A. McCloskey, Nucleic Acids Res. 1994, 22, 2183-96; H. Aurup, D. M. Williams, F. Eckstein, Biochemistry 1992, 31, 9636-41). Functionalized nucleotide triphosphates have been shown to be substrates for RNA polymerases (T. M. Dewey, A. Mundt, G. J. Crouch, M. C. Zyzniewski, B. E. Eaton, J. Am. Chem. Soc. 1995,117,8474-5; H. Aurup, D. M. Williams, F. Eckstein, Biochemistry 1992, 31, 9636-41).and catalytic RNA's dependent on the modified base for their activity have been selected (T. W. Wiegand, R. C. Janssen, B. E. Eaton, Chem. Biol. 1997, 4, 675-683; T. M. Tarasow, S. L. Tarasow, B. E. Eaton, Nature (London) 1997, 389, 54-57). Like RNA, DNA has also been selected to bind proteins and small molecules and more recently to catalyze reactions (S. E. Osborne, A. D. Ellington, Chem. Rev. (Washington, D.C.) 1997, 97, 349-370; M. Famulok, J. W. Szostak, Angew. Chem. 1992,104, 1001-11;Angew. Chem. Int. Ed. Engl. 1992, 31, 979-88; L. Gold, B. Polisky, 0. Uhlenbeck, M. Yarus, Annu. Rev. Biochem. 1995, 64, 763-97; R. R. Breaker, Chem. Rev. (Washington, D.C.) 1997, 97, 371-390; R. R. Breaker, Curr. Opin. Chem. Biol. 1997, 1, 26-31; J. R. Lorsch, J. W. Szostak, Acc. Chem. Res. 1996, 29, 103-10; R. R. Breaker, G. F. Joyce, Chem. Biol. 1994, 1, 223-9; B. Cuenoud, J. W. Szostak, Nature (London) 1995, 375, 611-14; R. R. Breaker, G. F. Joyce, Chem. Biol. 1995, 2, 655-60; C. R. Geyer, D. Sen, Chem. Biol. 1997, 4, 579-593; S. W. Santoro, J. G. F., Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 4262-4266; P. Burgstaller, 3M. Famulok, Angew. Chem. 1995,107,1303-06; Angew. Chem. Int. Ed. Engl. 1995, 34, 1189-92; D. Faulhammer, M. Famulok, Angew. Chem. 1996, 108, 2984-88; Angew. Chem. Int. Ed. Engl. 1996, 35,2837-2841; D. Faulhammer, M. Famulok, J Mol Biol. 1997, 269, 188-202; Y. Li, D. Sen, Nat. Struct. Biol. 1996, 3, 743-747; J. Burmeister, G. von Kiedrowski, A. D. Ellington, Angew. Chem. 1997, 109, 1379-81; Angew. Chem. Int. Ed. Engl. 1997, 36, 1321-1324; N. Carmi, L. A. Shultz, R. R. Breaker, Chem. Biol. 1996, 3, 103 9-1046; N. Carmi, H. R. Balkhi, R. R. Breaker, Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2233-2237).
While DNA possesses enhanced stability as compared to RNA, the lack of a 2'-hydroxyl group which provides for the enhanced stability of this molecule further reduces the functionality available to this molecule for chemistry. In contrast to the success achieved in identifying modified nucleotide triphosphates for RNA libraries, there is but a single example of a deoxynucleotide triphosphate, 5-(1-pentynyl)-2' deoxyuridine triphosphate, that has been demonstrated to be a good substrate for a thermostable DNA polymerase and utilized in an in vitro DNA selection study (J. A. Latham, R. Johnson, J. J. Toole, Nucleic Acids Res. 1994, 22, 2817-22). Indeed, difficulties in identifying modified deoxynucleotide triphophates substrates for the thermostable polymerases required for PCR have led recently to the development of novel strategies for in vitro selection without enzymatic amplification (J. Smith, E. V. Anslyn, Angew. Chem. 1997, 109, 1956-58; Angew. Chem. Int. Ed. Engl. 1997, 36, 1879-1881). These authors state that when modified oligomers are incorporated, the selection procedure is terminated after just one round of amplification since polymerases will not tolerate most modified mononucleotides. Therefore, the major impediment to the creation of novel functionally modified DNA catalysts and binding molecules is the determination of the substrate structures accepted by the thermostable polymerases.
The present invention provides a solution to this problem through the systematic synthesis of pyrimidine derivatives suitable for in vitro selections of modified DNAs.